Southern Blot
From ChoanoWiki
DAY 1
Run the gel:
− Run DNA that you wish to probe on an agarose gel. Pour the gel such that it is about 6mm thick
Depurination:
− Rinse the gel in dH20
− Place in a glass dish and cover with 0.25M HCl, shake gently for 30 minutes at room temp
− Pour off HCl and rinse gel with dH20
− Cover gel with denaturation solution, shake for 20 minutes
− Replace denaturation solution and shake for 20 minutes
− Pour off the denaturation solution and rinse with dH20
− Cover the gel with neutralization solution, shake for 20 minutes
− Replace the neutralization solution, shake for 20 minutes
The transfer:
− Cut 5 sheets of Whatman paper, one sheet of Zeta-Probe membrane and a 15cm stack of paper towels to the exact size of the gel
− Soak the membrane in dH20 for five minutes while setting up for the transfer
− Place sponge(s) in the bottom of a glass dish. The sponge(s) must be larger than the gel
− Place enough 10x SSC buffer in the transfer dish to immerse the sponge about half way up its side
− Place 3 sheets of Whatman paper on the sponge and flood the paper surface with buffer. Roll a pipette over the paper to remove bubbles
− Place the gel on top of the paper and remove any bubbles by pressing them away.
− Spread plastic wrap over the gel and cut out a window with a new razor blade allowing only the gel to emerge through the window.
− Flood the surface of the gel with buffer and place the pre-wetted membrane on top of the gel, being sure to line up the top of the gel with them membrane. Remove bubbles by rolling a pipette over the membrane.
− Flood the membrane with buffer. Wet 2 sheets of Whatman paper in 10x SSC and then place them over the membrane. Be sure they are aligned with the membrane and completely cover it without touching the gel.
− Stack the paper towels on the Whatman paper
− Do not put excessive weight on top of the gel
− Keep an excess of buffer in the dish but so not cover the top of the sponge
− Allow the transfer to continue overnight
DAY 2
− Separate the membrane from the gel, rinse briefly in 2x SSC and blot with filter paper.
− UV crosslink the DNA to the membrane and bake in the vacuum oven at 80 C for 30 minutes
Make the Aqueous pre-hyb solution:
1% SDS
5x Denhardt’s soln
5x SSC
100ug/ml denatured SSDNA
for 10mL:
1.0 mL 10% SDS
1mL 50x Denhardt’s
2.5 mL 20x SSC
100ul denatured SSDNA(10mg/ml)*
5.4 mL H20
- boil SSDNA for 10 minutes to denature and ice before adding to solution
Start blocking membrane:
− Wet membrane in 6x SSC
− Place membrane DNA-side-up in hyb tube and add 10ml of pre-hyb solution
− Incubate in hyb oven for 3 hours at 68 C
Labeling Probe:
Combine:
100ng DNA template
1ul random primers (125ng/ul)
add H20 to 30ul
− Boil at 100 C for 2 minutes
− Incubate on ice for 2 minutes
− Spin down quickly
− Return to ice
Add to the mix: 1ul 5mM dNTP solution
10ul 5x random priming solution
1ul 10mM DTT
5ul 10mCi/mL [α-32P] dCTP
2ul H20
1ul (5U) Klenow fragment
− Mix and spin
− Incubate at room temp for 1-2 hours
When labeling reaction is done: (about 45 minutes before the blocking is finished)
− Clean labeling reaction with Qiaquick Nucleotide Removal Kit ***be sure to dispose of radioactive waste properly!!!!
− Boil probe for 10 min
− Transfer to ice and spin down
− Add probe to pre-hyb
− Incubate overnight at 68 C
DAY 3
Wash membrane:
- remember to discard all radioactive solutions in waste!!!!
- all washes done in hyb oven with rotation
− Prewarm 0.2x SSC, 0.1% SDS at 42 C and 0.1x SSC, 0.1% SDS at 68 C
− Pour off hyb solution
− Add 10mL 2x SSC, 0.1% SDS and incubate for 5 min at room temp
− Change wash solution and repeat
− Replace wash with 10mL 0.2x SSC, 0.1% SDS and incubate for 5 min
− Change wash solution and repeat
− Survey membrane with the geiger counter for background
− If needed, add 10mL 0.2x SSC, 0.1% SDS prewarmed to 42 C and incubate for 15 min at 42 C
− Change wash solution and repeat
− Survey membrane with the geiger counter for background
− If needed, add 10mL 0.1x SSC, 0.1% SDS prewarmed to 68 C and incubate for 15 min at 68 C
− Change wash solution and repeat
− Remove the final wash and rinse membrane in 2x SSC at room temp
− Blot excess liquid and cover with plastic wrap
− Perform autoradiography
Solutions:
IM Tris-Cl, pH 7.0
121.1g Tris base
800mL dH20
− Let solution come to room temp
− Adjust pH to 7.0 with HCl
− Adjust volume to 1L
0.25M HCl (for depurination)
20.9mL 12N HCl (37%)
979.1mL dH20
1L total
Denaturation soln.
20g NaOH
87.6g NaCl
1L total
Neutralization soln.
500mL 1M Tris-Cl
87.6g NaCl
1L total
20X SSC, pH7.0
175.3g NaCl
88.2g Sodium Citrate
800mL dH20
− Adjust pH to 7.0 with HCl
− Adjust volume to 1L
